Multi-Segment Direct Inject nano-ESI-LTQ-FT-ICR-MS/MS For Protein Identification.

Reversed phase high performance liquid chromatography (HPLC) interfaced to electrospray tandem mass spectrometry (MS/MS) is commonly used for the identification of peptides from proteolytically cleaved proteins embedded in a polyacrylamide gel matrix as well as for metabolomics screening. HPLC separations are time consuming (30-60 min average), costly (columns and mobile phase reagents), and carry the risk of column carry over between samples. The use of a chip-based nano-ESI platform (Advion NanoMate) based on replaceable nano-tips for sample introduction eliminates sample cross-contamination, provides unchanging sample matrix, and enhances spray stability with attendant increases in reproducibility. Recent papers have established direct infusion nano-ESI-MS/MS utilizing the NanoMate for protein identification of gel spots based on full range MS scans with data dependent MS/MS. In a full range scan, discontinuous ion suppression due to sample matrix can impair identification of putative mass features of interest in both the proteomic and metabolomic workflows. In the current study, an extension of an established direct inject nano-ESI-MS/MS method is described that utilizes the mass filtering capability of an ion-trap for ion packet separation into four narrow mass ranges (50 amu overlap) with segment specific dynamic data dependent peak inclusion for MS/MS fragmentation (total acquisition time of 3 minutes). Comparison of this method with a more traditional nanoLC-MS/MS based protocol utilizing solvent/sample stream splitting to achieve nanoflow demonstrated comparable results for protein identification from polyacrylamide gel matrices. The advantages of this method include full automation, lack of cross-contamination, low cost, and high throughput.